The present invention relates broadly to the isolation, identification and use of natural products. More particularly, this invention relates to substantially pure forms of two related biologically active diterpenoid compounds that are useful to regulate the development of microorganisms. Specifically, these substances may be used to induce conidiation in, and thereby also to inhibit the growth of, such microorganisms.
The continuous deposition of new cell wall and plasma membrane material at a fixed cellular site (termed xe2x80x9capical extensionxe2x80x9d) is a successful growth pattern which is widely observed in prokaryotic and eukaryotic organisms (Heath, 1990). The derived tubular structure (the hypha) offers great practical advantages for the colonization of solid substrates, as well as the undertaking of fungal infection. It acts as a device providing sheer physical force combined with a versatile arsenal of apically secreted enzymes directed at the hydrolysis of biopolymers and other bioconversions. However, apical extension growth is unsuitable for dispersive purposes or resistance to unfavorable environmental conditions, and filamentous microorganisms have evolved alternative morphogenetic programs to generate cell forms which cater to these requirements: spores (Smith and Berry 1974).
The formation of conidia or asexual spores among filamentous fungi is a complex process in which a succession of precisely regulated cellular events take place in tight coordination. Environmental or endogenous stimuli are thought to trigger a set of genetically determined programs which lead to the cessation of apical growth, and to the development of reproductive asexual structures which finally result in the formation of conidia.
Significant advances in the knowledge of the genes controlling conidial development have been carried out in the last two decades, using mainly Aspergillus nidulans as model organism. Three regulatory genes: brlA, abaA and wetA (Clutterbuck, 1969; Mirabito et al., 1989) are involved, and brlA (coding for a transcription factor) occupies a central position in the control of development. Its expression is sufficient (and therefore necessary) for the initiation of conidial development in Aspergillus nidulans (Adams et al., 1988). Besides these, other early acting genes have been identified, which are necessary for the activation of brlA: flbA, flbB, flbC, flbD, flbE and fluG (Wieser et al., 1994). However, little is known about the factors which trigger the process of conidial development and the mechanism that is followed before the first gene is activated. It is suspected that an environmental stimulus or set of stimuli are perceived by the fungus, leading to the triggering of a signal transduction system which, in turn, activates the conidiation program.
The environmental stimuli reportedly implicated in the induction of conidiation have been a matter of investigation since the pioneering works of Sir Hans Klebs (1896). They include: emergence to the atmosphere, nutrient depletion (especially the nitrogen source), presence of cations, temperature, light stimulation and pH changes in the medium. However, the precise way in which these diverse stimuli influence the process in different fungi, or their mechanism of action, remains in the realm of hypothesis.
Without doubt the most widespread and powerful stimulus leading to conidiation is the emergence to the aerial environment (Morton, 1961) and several mechanisms have been proposed to explain what happens in this case (oxidative stress and desiccation). However, no definitive demonstration of the validity of one or more of these mechanisms has emerged. In any case, changes in surface characteristics of hyphae are known to occur on emergence (Morton, 1961), which could be related to the induction mechanism, but no modern studies on this effect have been pursued.
Contrary to the widespread subaerial conidiation among filamentous fungi, conidiation in submerged culture is a relatively rare event in fungal cultures, and is difficult to induce through manipulations of the culture conditions in the best cases (Morton, 1961; Galbraith and Smith, 1969). Controlled nutrient limitation or the addition of special agents which have been empirically shown to function as inducers is a common strategy. One such example is the addition of calcium ions.
Fungi belonging to the genus Penicillium respond to the addition of calcium in liquid culture with a precisely timed morphogenetic program leading to conidiation (Foster et al., 1945, Hadley and Harrold, 1958; Ugalde and Pitt, 1983; Pascual et al., 1997), in a way indistinguishable from that occurring spontaneously in subaerial culture. The mode of action of the cation, as well as the changes taking place at the cellular level as a result of its action, have been extensively studied for the last twenty years. The results of this research support the view that the mode of action of the cation is unrelated to entry and subsequent rise in cytosolic calcium levels, but is mediated through an interaction taking place at the cell surface.
Conidia produced by filamentous fungi such as those belonging to the genus Penicillium and to other genera as well can be harvested and employed in various useful ways. For example, such conidia can be employed in fungicides, herbicides and insecticides, and they can also be employed as biotransformation agents or as catalysts in chemical reactions; they are also useful as additives in the food industry since, for example, strains of the genus Penicillium are used in the production of Roquefort cheese and Danish blue cheese, and strains of the genus Aspergillus are widely used in fermentation processes for the production of enzymes and fermented foods. Thus, further elucidation of the mechanisms leading to conidiation, and of the events and molecules that trigger it, would have potential commercial significance in several major industries.
Moreover, since the formation of conidia leads to (or occurs in tandem with) the cessation of apical growth, further elucidation of the mechanisms leading to conidiation, and of the events and molecules that trigger it, would have potential biomedical significance as a means of fungal control. In particular, the isolation and purification of the molecular substances that induce conidiation would be useful as a means of inhibiting or even preventing invasive and/or infectious fungal growth, and also could potentially lead to the design of analogs of such molecules which, conversely, could be used to inhibit conidiation and thereby stimulate or even enhance fungal growth. However, the prior art is deficient in any description of such biologically active (or xe2x80x9cbioactivexe2x80x9d) substances.
It is therefore an object of the present invention to fulfill a long-standing need and desire in the art and to provide bioactive substances having conidiation-inducing activity.
Briefly, the invention relates to substantially pure forms of hitherto unknown secondary metabolites of microorganism strains belonging to the genus Penicillium, and likely to other genera as well. These metabolites are tetracyclic diterpenoid compounds, and in one embodiment of the present invention there are provided tetracyclic diterpene compounds isolated from Penicillium cyclopium having the following general structural formula (I): 
wherein R stands either for xe2x80x94OH (hydroxyl) or xe2x95x90O.
In another more specific embodiment of the present invention there is provided a compound isolated from Penicillium cyclopium having the following structural formula (1): 
In yet another more specific embodiment of the present invention there is provided a compound isolated from Penicillium cyclopium having the following structural formula (2): 
Spectral data obtained for compounds (1) and (2) were in agreement with the structure shown in formula (I). It was found that these compounds efficiently induce conidiation not only in the producer microorganism P. cyclopium, but also in other members of the genus Penicillium and outside that genus as well. This invention also includes the acylated derivatives of the compound having the structural formula (I).
In still other embodiments of the present invention, there are provided methods for producing, as well as methods for using, the compounds disclosed herein. In general, the method of producing these bioactive substances comprises culturing, under aerobic conditions, a strain of microorganism belonging to the genus Penicillium (or a mutant or variant thereof) capable of elaborating said bioactive substances, in a suitable nutrient medium containing at least a carbon source and a nitrogen source, at a pH and temperature, and for a time, sufficient for production of said bioactive substances, and thereafter accumulating said substances in the culture medium and isolating said substances therefrom.
The methods of use generally comprise the application of these bioactive substances, preferably in combination, as conidiation inducers, to at least one strain of microorganism, preferably one belonging to the genus Penicillium or the genus Aspergillus; the methods include any related biochemical, microbiological and clinical effects associated with the morphogenetic process, such as growth inhibition, whether or not the morphogenetic process is completed.